UNIVERSITY OF YORK BIOLOGY. Developmental Biology

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1 Examination Candidate Number: UNIVERSITY OF YORK BSc Stage 2 Degree Examinations Department: BIOLOGY Title of Exam: Developmental Biology Desk Number: Time allowed: 1 hour and 30 minutes Total marks available for this paper: 80 This paper has three parts: Section A: Short answer questions (30 marks) Answer all questions in the spaces provided on the examination paper Section B: Problem questions (20 marks) Answer all questions in the spaces provided on the examination paper Section C: Long answer question (marked out of 100, weighted 30 marks) Answer either question A or question B Write your answer on the separate paper provided and attach it to the back of the question paper using the treasury tag provided Instructions: The marks available for each question are indicated on the paper A calculator will be provided For marker use only: Total as % DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR page 1 of 13

2 SECTION A: Short answer questions Answer all questions in the spaces provided Mark total for this section: 30 Section A: 1. a) What evidence suggests that Bicoid acts a morphogen? (3 marks) Answer: Bicoid protein forms an anterior to posterior gradient in the early fly embryo (1 mark). When a fly is genetically engineered to have multiple copies of the bicoid gene the levels of Bicoid protein are higher and diffuse to more posterior regions(1 mark). The threshold level of Bicoid at which the Hunchback gene is activated is present more posteriorly and/ or the evidence is that in these flies with extra Bicoid, Hb is expressed further posteriorly(1 mark). FEEDBACK: you needed to describe an experiment and interpret it. Also mention gradient of protein. Many people just describe what is known about bicoid, even did this really well, but I was asking you to tell me the evidence: how do we know this?. b) What evidence suggests Shh acts as a morphogen? (3 marks) Answer: Shh protein diffuses from the floorplate of the neural tube (1 mark). When intermediate neural plate explants are exposed to different concentrations of Shh protein in culture, floorplate and motor neurons form in response to high concentrations of Shh and more dorsal interneurons form in lower concentrations of Shh (1 mark). The neuronal precursors that are induced at high-to-low conc Shh is consistent with the in vivo position of these precursors in a ventral-to-dorsal position in the neural tube (1 mark). Students might use the example that a bead of Shh will induce digit 4 at high concentration and digit 2 at low concentration (1 mark). FEEDBACK: you needed to describe an experiment and interpret it. Also mention gradient of protein. c) State the molecular nature of these two morphogens. (1 mark) Answer: Bicoid is a transcription factor; Shh is a secreted/diffusible signalling molecule (1 mark) page 2 of 13

3 For full marks in this Q, the answer must at some point (in answering a, b, or c) define a morphogen as a substance to which cells respond differently to at different concentration. Relation to MLOs: Describe experimental evidence that supports the key concepts presented in the module 2. Describe the evidence that shows FGF signalling promotes the proximal-distal outgrowth of the vertebrate limb bud. (3 marks) Answer: FGF8 and FGF4 are expressed in the AER (1 mark). The AER is essential for PD outgrowth, when it is excised PD outgrowth stops. This can be rescued with an FGF soaked bead (1 mark). An FGF bead implanted into the flank of an early chick embryo will induce the formation of an ectopic limb (1 mark). FEEDBACK: you needed to describe an experiment and interpret it. Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups 3. a) What does our knowledge of Pax6 function in modern protostomes and deuterostomes tell us about the anatomy and development of urbilateria? (2 mark) Answer: Functional studies in both groups indicate a role for Pax6 in regulating the development of light sensing organs. This indicates that urbilateria, which was the last common ancestor of protostomes and deuterostomes, probably also had a light sensing organ and that the development of this organ was regulated by a Pax6-related gene. FEEDBACK: very good on this Relation to MLOs: Describe experimental evidence that supports the key concepts presented in the module b) The genome of urbilateria is believed to have contained a single Hox gene cluster. Explain the rationale for this conclusion. (1 mark) Answer: The genomes of modern protostomes and primitive deuterostomes all contain a single Hox cluster. This suggests that the page 3 of 13

4 last common ancestor also had a single cluster. FEEDBACK: needed to refer to last common ancestor of Prots and Deuts Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups c) The posterior segments of primitive arthropods frequently bear appendages. In contrast, the abdominal segments of insects do not develop appendages. What is the molecular basis for this difference in body plan? (2 marks) Answer: Expression of the distaless gene is required for the growth of arthropod limbs (1 mark). The Ubx gene is expressed in the posterior region of both primitive arthropods (crustacea or velvet worms) and abdomen of insects. However, during insect evolution ubx has acquired the ability to repress distaless expression and therefore the development of limbs in the abdomen (1 mark). FEEDBACK: very good on this Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups. 4. a) What is the molecular basis of neural induction by noggin, chordin and follistatin in explants of amphibian ectoderm? (3 marks) Answer: The default developmental pathway for ectodermal cells is neural (1 mark). However, low level BMP signalling in the amphibian ectoderm activates the expression of genes required for the epidermis and diverts ectodermal cells from this default neural pathway (1 mark). Noggin, chordin and follistatin induce neural tissue through binding to extracellular BMPs and inhibit their ability to activate BMP receptors and epidermal gene expression (1 mark). FEEDBACK: you needed to say that the antagonists bind BMP to stop it binding its receptor: molecular mechanism Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups. page 4 of 13

5 b) What is the experimental evidence that noggin, chordin and follistatin have overlapping and semi-redundant functions in neural induction during normal amphibian development? (2 marks) Answer: Individual or even double morpholino mediated knockdowns of noggin, chordin and follistatin have little effect on neural development (1 mark). However, there is a catastrophic loss of neural development when all three are knocked down (1 mark). FEEDBACK: ALL of you got this. It must have been taught v well. Although more than half of you spell triple as tripple...kind of funny. No marks off for that. Relation to MLOs: Describe experimental evidence that supports the key concepts presented in the module. 5. What does the phenotype of the phantastica mutant reveal about leaf development? (4 marks) phan mutant phenotype reveals the importance of the dorsoventral axis and reveal the radial axis of the leaf (1 mark). Radialised leaves of the phan mutant lack a dorsoventral axis and fail to expand laterally (1 mark). This demonstrate that there is an interaction between upper/adaxial/dorsal tissue and lower/abaxial/ventral tissue. The juxtaposition of these tissues stimulates a novel axis of growth (1 mark) as is seen in other phan mutant leaves. Petals of phan mutants also show reduced dorsoventrality indicating that all lateral organs are determined by the same developmental axes (1 mark). 6. What evidence indicates that the interaction between STM and AS1 is conserved in Arabidopsis thaliana and Cardamine hirsuta (3 marks) In both Arabidopsis and Cardamine these genes have mutually exclusive expressions patterns (1 mark). as1 mutants have the the same morphological phenotypes in both species and show overexpression of STM and other KNOX genes (2 marks) 7. a) What is the function of the REDUCED COMPLEXITY ( RCO ) gene in Cardamine hirsuta? (1 mark) RCO restricts growth at leaf margins to produce leaflets in C. hirsuta. page 5 of 13

6 The rco mutant lacks leaflets, unlike the wild-type leaf which has leaflets. RCO is part of a cluster of three closely related genes in Cardamine hirsuta that includes LATE MERISTEM IDENTITY1 ( LMI1 ). b) What do we know about the expression pattern of LML1 in Cardamine hirsuta? (1 mark) LMI1 has a complementary pattern of expression compared to RCO (1 mark). RCO is expressed towards the base of leaflets developing in a more proximal position in the leaf and LMI1 is expressed towards the tips of leaflets developing in a more distal position. c) What would be the phenotype of an rco mutant constitutively expressing the LML1 gene? (1 mark) It is likely that the constitutive expression of LMI1 would rescue the rco phenotype. (1 mark) Although RCO and LMI1 appear to have different roles in leaf development because the have a different temporal and spatial expression patterns, the coding regions of these genes are very similar suggestions that they have the very similar functional roles. SECTION B: Problem questions Answer all questions in the spaces provided Mark total for this section: members of the Wnt family of secreted signalling molecules are expressed in Xenopus development. The downstream transcriptional effectors of Wnt signalling are TCF transcription factors, which bind to Wnt response elements with a consensus sequence ATCAANG (where N=A, C, G or T) in the enhancers of Wnt regulated genes. Figure 1A page 6 of 13

7 Figure 1B Expression of the wix1 gene is upregulated in isolated Xenopus embryonic cells in response to wnt8 protein. To study the transcriptional regulation of wix1, a region of the gene upstream of the transcriptional start site was isolated and sequenced (Figure 1A). This was used to make a wix1 luciferase reporter gene (wix1-luc) (Figure 1B). Figure 2 Figure 2A shows a plot of the relative levels of expression from the endogenous wix1 gene in the developing embryo during the first 16 hours of development, as determined by quantitative PCR. Figure 2B shows a plot of luciferase activity in Xenopus embryos transgenic for the wix1-luc reporter over the same period. a) What do these data tell us about the wix1-luc transgene and the upstream sequence that it contains? (3 marks) Answer: The temporal expression profile for the endogenous gene is closely matched by that of the reporter transgene (1 mark). This indicates that the reporter recapitulates the expression of the endogenous gene. Therefore, the identified upstream region of wix-1 within the wix1-luc reporter probably contains all the regulatory information necessary to drive the normal page 7 of 13

8 expression pattern of this gene (2 marks). FEEDBACK: Many people saw that the expression of the reporter match the mrna profile and pointed out this means all regulatory regions required for normal expression must be in the reporter well done. Additional wix1-luc derived constructs were produced, in which either bases 1 to 30 (wix1-d30-luc) or bases 31 to 54 (wix1-d54-luc) were deleted from the upstream region of wix1. Figure 3 shows the activity of the wild-type and deletion wix1-luc reporters in control embryonic cells and cells treated with wnt8. Figure 3 b) Based on the available data, present a hypothesis to explain the observed effects on reporter activity. (4 marks) Answer: Examination of the sequence 31 to 54 reveals the presence of 2 consensus TCF binding sites for TCF transcription factors (2 marks). These are required for mediating the reporter response to Wnt signaling. Removal of these sites renders the reporter unresponsive to Wnt signalling (2 marks). FEEDBACK: Why why why do so many of you think Wnt8 (a signalling molecule) can bind DNA??? We even tell you TCF is the effector of the Wnt8 signalling pathway...but still 80% of you say that Wnt8 binds DNA. Also only a few of you thought to look at the DNA sequence and spot the 2 consensus sequences!! c) Subsequently, wix1-luc activity was compared in control embryos and embryos injected with morpholino oligos directed against the translation start site of wnt8. No difference was noted in luciferase activity between the two groups. What might explain this observation? (3 marks) Answer: The most obvious answer is the fact that there are multiple wnts expressed in early Xenopus development. There is likely to be some page 8 of 13

9 functional redundancy between these genes (2 marks). Therefore, knocking down only one wnt down may not have an effect on the activity of the wix1-luc reporter. FEEDBACK: only a few clever people thought about the other 15 Wnts you were told about in the question intro! Well done to you. Also credit to those who considered stored maternal protein. Relation to MLOs: Interpret novel experimental evidence related to topics covered in the module. 9. Whilst working with Arabidopsis halleri, a close relative of Arabidopsis thaliana, you decide to investigate the function of STM -like genes in this species. After an initial search, you sequence three genes that appear to be strong candidates for the Arabidopsis halleri STM gene. a) How would you determine when and where these STM -like genes are expressed in Arabidopsis halleri? (3 marks) The simplest way to derive this information is to carry out in situ hybridisations. (a full description about an in situ etc. would gain full marks) Other experiments might also provide the same information such as promoter-gus fusions using the promoter region from the STM -like genes from Arabidopsis halleri. Answers might also suggest using the KN1 antibody to determine the locate of STM-like proteins via an immunolocalisation. This approach is useful as it has been demonstrated that this antibody does recognise the broader family of STM-like genes in plants. Furthermore, the KN1 transcript is found in a broad domain of cells than it is expressed in indicating that is worthwhile comparing gene expression with protein localisation. (full alternative answers would also get full marks) b) Where in Arabidopsis halleri would you expect the equivalent gene to STM to be expressed? (2 marks) page 9 of 13

10 You would expect the equivalent gene to have the same expression pattern as STM in Arabidopsis, although some closely related genes have similar patterns of expression it is likely that is these Arabidopsis species that the most equivalent genes would have similar patterns of expression in central region of the shoot apical meristem and possibly in developing leaf primordia (2 marks). c) How would you confirm that one of the genes from Arabidopsis halleri is functionally equivalent to STM from Arabidopsis thaliana? (5 marks) There are two main ways to do this. The first is to knockout each gene and compare the mutant phenotypes (2 marks), each should be rescued by overexpressing the equivalent gene in a different species (3 marks). page 10 of 13

11 SECTION C: Long answer question Answer one question on the separate paper provided Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B or C. Mark total for this section: 30 EITHER A) With reference to experimental evidence, describe the conserved pathway that regulates neurogenesis in bilaterally symmetrical animals. Answer outline: Would be useful to have definition of neurogenesis, also where and when it occurs in flies and frogs Importance of proneural genes coding for bhlh transcription factors in defining proneural clusters as regions competent to form neurons Experimental evidence from fly mutants and overexpression in frogs Importance of lateral inhibition mediated by notch delta signalling in picking out cells to differentiate. Experimental evidence from fly mutants and overexpression frogs. Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups. Describe experimental evidence that supports the key concepts presented in the module. Feedback: Most answers addressed the key regulatory interactions between proneural and neurogenic genes with some accuracy. However, many answers were let down by not giving the pathways context in terms of evidence from flies and frogs. This is important to highlight the conserved features and where appropriate page 11 of 13

12 differences. Similarly with the experimental evidence there was a tendency to be too general and not discuss what species the experiments were relevant to. There was generally a lack of accuracy in describing the experimental detail. I got the impression in some cases candidates were not that aware of the experiments but were using their general understanding of the regulatory pathways to predict (usually accurately) the outcomes of hypothetical experiments that haven t been done! OR B) Evaluate the evidence that vertebrate Hox genes are functionally conserved orthologues of the Drosophila homeotic genes. Answer outline: Clustering in genome in flies and mice, although mice have 4 Hox clusters and flies only one. Co-linear expression in flies and mice (3-5; A-P). Expression boundaries at borders of cell lineage restriction (parasegment boundaries and rhombomeres). Loss of function mutants in flies have clear effects of PS identity; posterior to anterior transformations (definitive homeotic transformation). Mice knockouts, B4 for instance, show a milder phenotype but some signs of A-P transformation in the vertebrae. Discuss redundancy of paralogous Hox genes. Relation to MLOs: Describe and discuss, with reference to specific examples, the highly conserved mechanisms regulating the development of body plans and organ systems in a range of animal and plant groups. Describe experimental evidence that supports the key concepts presented in the module. FEEDBACK: Some really good answered stayed focussed on addressing conserved function, which needed to include gene targeting Hox genes in mice as well as what is learned about function from Drosophila mutants. page 12 of 13

13 OR C) Evaluate the evidence from Arabidopsis and Antirrhinum that explains the evolutionary history of the c-function genes required for floral organ identity and development in flowering plants. An explanation of the evidence that both genes are derived from a common ancestor that existed about 150mya An explanation of the evidence a subsequent gene duplication event initiated the separate PLENA and AGAMOUS lineages An explanation of the evidence that following subfunctionalisation, alterations to gene expression patterns and a whole genome duplication event in Arabidopsis distinct roles evolved for PLENA and FARINELLI in Antirrhinum and AGAMOUS, SHATTERPROOF1 and SHATTERPROOF2 in Arabidopsis An explanation of the evidence that AGAMOUS retains the c-function in Arabidopsis and whilst PLENA retained the c-function in Antirrhinum. page 13 of 13